Adeno-Associated Virus type 2 (AAV-2) is unique among animal viruses in its ability to establish a latent infection by integrating site-specifically into a locus of chromosome 19. Moreover, because of its apparent non- pathogenicity, broad cell and tissue tropism and ability to infect in a cell-cycle independent fashion, AAV has emerged as one of the most promising vectors for gene therapy. A number of studies indicate that AAV- mediated site-specific integration requires only three players: a) the AAV Rep78 or Rep68 non-structural proteins, b) an AAV DNA element containing a Rep Binding Site (RBS) and c) the cellular AAVS1 integration site. During a decisive event, these players come together to form a protein-DNA complex that serves as the starting point for an integration process that is considered to be parallel to the AAV replication initiation process. This pre-integration complex is dependent on the ability of Rep68/78 proteins to bind the integration site AAVS1, promote its melting and catalyze a strand-specific nicking reaction that leaves a Rep68/78 protein covalently bound to the integration site. Although a general view of this mechanism has been achieved through the study of AAV DNA replication, little is known regarding the architecture of the Rep-AAVS1 complex and the steps that lead to its formation. The long-term goal of this proposal is to establish a structural, biophysical and biochemical framework required to understand the mechanism of AAV-mediated site-specific integration. Our approach is to couple structural studies using X-ray crystallography, cryo-EM and small angle X-ray scattering (SAXS) with biophysical, kinetic and functional studies to get a complete understanding of the events that lead to site-specific integration. We expect that the results obtained in this research proposal will serve both as a starting point to generate accurate models to understand this unique mechanism and also will give us the knowledge and tools to potentially design Rep proteins with novel specificities that could be used to develop new technologies in the gene therapy field. PUBLIC HEALTH RELEVANCE: This study will contribute significantly toward understanding the mechanism of AAV mediated site-specific integration. In addition, it will contribute to our understanding of origin DNA replication initiation. Knowledge gained from this proposal will serve as the basis for designing novel gene therapy vectors that could be targeted to a specific region in the chromosome.